This invention relates to a method and apparatus for measuring certain parameters of particles and then optionally sorting the particles according to the values of the parameters measured, especially biological cells or portions of biological cells that are measured and sorted.
A cell sorter is an instrument that physically separates cells according to certain parameters. Many cell sorters use techniques to distinguish subpopulations of cells by employing a unique blend of modern technologies such as fluidics, electric fields, lasers, optics, analog and digital electronics, and computers and software. These techniques are often referred to as flow cytometry.
In a typical, conventional use of flow cytometry, selected cells are labeled with fluorescent molecules that bind specifically to the constituent(s) (e.g. using a fluorescently-labelled antibody to a particular surface antigen) to be measured. Certain, desired cells (a subpopulation of cells) are fluorescently labeled while other cells in the sample of cells are not fluorescently labeled, such that the fluorescently labeled cells may be selectively identified from a heterogeneous cell population. These fluorescently labeled cells are contained within the sample of cells that will be measured and sorted. Referring to FIG. 2, the sample of cells in a stream of a saline solution and a cell-free sheath fluid are supplied to a droplet generator 23 by cell sample inlet 23c and sheath fluid inlet 23d respectively. The sheath fluid confines, by hydrodynamic focusing, the sample of cells to a central core of the laminar flow that is leaving the droplet generator at orifice 23a. Before droplet generator 23 actually forms droplets, the combined stream of sheath fluid and labeled cells flows through a measuring region in an illumination frame which holds the droplet generator.
Inside the measuring region, the cells in the sample of cells pass, typically one by one, through a beam of excitation light from a light source 21 (e.g. a laser or arc lamp), such that each fluorescently labeled cell produces a short flash of fluorescence when passing through the light beam, the intensity of which is proportional to amount of the fluorescent label on the cell. These flashes of fluorescence are collected by a fluorescence collection lens 24b, which focuses the light on a sensitive fluorescence detector 24a. Detector 24a transforms the flashes of light into electrical pulses, which are measured and recorded by electronics 24c and optionally a computer 24d. Each cell also causes scattering of the excitation light at least in the case where the light source is a laser. The pattern of this scattering is a function of the size, shape and structure of the cell. The resulting flash of scattered light is recorded by a light scattering detector 24f and electronics 24e. Multiple fluorescence detectors may be used to detect several different fluorescent conjugates bound to the same or different cells in order to further distinguish different cell types in a heterogeneous mixture. Thus, multiple parameters including fluorescence at different wavelengths, as well as size and shape or structure, are recorded for each individual cell in the sample of cells.
Once these measurements are made, the cell sorter has the ability to selectively remove certain cells from the jet of sheath fluid. Just before the droplet-separation point, which occurs shortly after the cells leave the nozzle having the orifice 23a which is typically at the bottom of droplet generator 23, the droplets containing selected cells become selectively charged by a charging pulse 25a which is applied to the conductive fluid carrying the sample cells into the droplet generator 23. The charging pulse 25a is produced by a charging system control logic 24g which is coupled to receive signals from electronics 24c and electronics 24e; these signals indicate whether the cell, which was measured in the measuring region of the jet intersected by the beam of light from the light source 21, is a selected cell which is to be charged. The charging system control logic 24g analyzes these signals and determines whether the cell is to become a selected cell and, if so, applies the charging pulse to the conductive fluid carrying the cells just before the droplet carrying the cell breaks off of the jet of fluid. The jet leaving the orifice 23a is a substantially continuous stream of conductive fluid which applies the charging pulse, typically applied near the top of the droplet generator, to reach the droplet which is about to fall off of the jet below the orifice. The droplet carrying the selected cell separates as a charged droplet from the jet and falls, under the force of gravity, through a constant electric field produced by the deflecting system 26. If the cell that was measured is not a selected cell, then charging system control logic 24g will not apply the charging pulse, and the droplet containing this cell will separate from the jet as an uncharged droplet and will fall through the electric field largely unaffected by this field. Typically, one droplet will contain only one cell so that the selective charging of a droplet will select only one cell. The droplets are formed in the conventional manner by the ultrasonic vibrations of the ultrasonic transducer 23b which is coupled to droplet generator 23 which is supported by the illumination frame.
The charged and uncharged droplets then pass through a constant electrostatic deflection system 26 typically having a negative and a positive deflecting plate. Deflecting system 26 alters the trajectory in which the charged droplets are traveling such that the charged droplets are physically separated from the uncharged droplets according to the value of the parameters measured by the electronics. After passing through deflecting system 26, the droplets are collected in a cell collector 27 which may have several different collection receptacles. For more general background information refer to Flow Cytometry and Sorting, Second Edition by Myron R. Melamed, Tore Lindmo, Mortimer Mendelsohn, published by Wiley-Liss NY, NY, 1990. Also see, for example, U.S. Pat. No. 5,150,313, and U.S. Pat. No. 3,560,754.
Cell sorters have the ability to measure several parameters of each individual cell to determine the size, structure, and the precise contents of various cellular constituents. Today, flow cytometers can measure cells and other particles all the way down to submicroscopic sizes, that is, to approximately 0.1 .mu.m and have sensitivities sufficient to detect 10.sup.-18 grams of a specific substance per cell. The ability to make multiple measurements on each cell, together with the resolution and sensitivity attainable with such measurements in cell sorters, makes possible the isolation of cell subpopulations having a purity and specificity of function that can be obtained in no other way.
Cell sorters are used in various fields of biology and medicine, including cell-cycle studies in relation to effects of drugs and radiation, immunology, ploidy determination in cancers, and studies of cellular parameters. For example, a flow cytometer can readily distinguish between different phases of the cell cycle in asynchronously growing cell cultures or can discriminate between different subsets of lymphocytes in immunology.
Although technology has led the way for a new generation of cell sorters that are simpler to operate and maintain, and significantly less expensive both to purchase and to operate, the inability to measure and sort cells in a sterile environment significantly limits the types of applications for the cell sorter. While small enclosures around the nozzle, droplet generator, electrostatic deflection plates, and collection receptacles have been suggested and used to protect users of the cell sorter from aerosols (generated around the nozzle) containing hazardous materials, these enclosures have not maintained the sterility of the cell stream before, during and after the sorting of the cells. The development of a cell sorter which is reliable, easy-to-use, and economical, and capable of operating in a sterile manner is necessary to achieve more widespread acceptance of the cell sorter, in both the clinical and research uses. Moreover, the enclosed environment protects the user of the flow cytometer from unsafe contaminants or cells which may be in the sample being measured and optionally sorted.